ABSTRACT While advancing age is a major risk factor of Alzheimer?s disease (AD), it is unclear why the incidence of AD rises dramatically as we grow older. This proposal will develop a powerful human induced pluripotent stem (iPS) cell resource to advance our understanding of the impact of aging on the development of AD cell pathology; particularly on the interplay between cellular aging, amyloid precursor protein (APP) and trisomy 21. Our studies will capitalize on an existing isogenic panel of iPS cells derived from a Down Syndrome (DS) patient, but will also include non-DS AD patient-derived iPS cells. Early-onset AD dementia is almost inevitable in individuals with DS, who notably also exhibit premature aging. Essentially all individuals with DS have amyloid brain pathology beginning as early as adolescence. Our preliminary results and other published evidence indicate that neurons differentiated from DS patient-derived iPS cells exhibit multiple hallmarks of AD cell pathology and provide a robust human cell model to study AD pathogenesis. The Lawrence lab has created a well characterized, all-isogenic panel of DS patient-derived cell lines, some of which are engineered to silence one Chr21 by inducible expression of XIST RNA, and others are normal disomic due to spontaneous loss of one Chr21. To build on this foundation to study the impact of aging, the Zhang lab, which has long studied cellular senescence and aging, will join forces with the Lawrence lab to induce cellular aging in multiple AD iPS cell lines by two different approaches. Using the CRISPR/Cas9 technology, we will target two important regulators of cellular aging, telomerase and Klotho. Several AD cell pathologies (A?42 secretion, amyloid aggregates, tau phosphorylation, 4R-tau production, endosome enlargement and cell loss) will be examined in iPS cells differentiated to cortical neurons, glia and other neural cells in 3D organoid cultures. In addition to DS, multiple familial or sporadic AD iPS cell lines will be studied through collaboration with Tracy Young-Pearse (Harvard Medical School). Aim 1 will target telomerase to produce iPS cells which undergo telomere shortening, a characteristic of aging cells. These cells become poised for cellular senescence but only after substantial proliferation, allowing us to address the impact of this form of cellular aging in differentiated neural cells. Aim 2 will manipulate the expression of Klotho, deficiency of which is strongly linked to aging and neurodegeneration in mice, and increased expression of which mitigates cognitive decline in a mouse model of AD. Further, we will test a concept of using Klotho protein with potential to mitigate aging effects on AD pathogenesis. This research has high potential to produce broadly valuable cellular resources as well as address several specific hypotheses that will advance our understanding of the interplay between aging and AD pathogenesis.